The overall aim described in this proposal is to characterize the complete set of transfer RNA genes for a chloroplast DNA. Chloroplast genomes are generally believed to encode all of the tRNAs involved in chloroplast protein synthesis. Since chloroplast DNAs are relatively small, and can be isolated intact, it is a feasible goal to locate and characterize every chloroplast tRNA gene, and to determine the anticodon and cognate amino acid for each. The chloroplast genome chosen as most amenable for this study is that of Euglena gracilis. We plan to map the location of the tRNA genes with respect to the known restriction nuclease cleavage map for this genome by using hybridization of chloroplast tRNAs to membrane filter blots of chloroplast DNA restriction fragments. Cloned segments of DNA will be used for detailed analysis and mapping, to determine the precise location of tRNA genes, and to test if multiple genes are clustered into transcription units and co-transcribed. Our goal is to identify tRNA coding loci, to determine patterns of gene organization, and to study the nature of the primary tRNA transcript for as many tRNA genes as can be identified. We also propose to begin a systematic analysis of the DNA sequence of tRNA genes, and gene clusters if present, as well as the adjacent regions that may be involved in gene regulation. A long-range aim is to initiate studies on the regulation of tRNA gene expression during chloroplast biogenesis, and to determine if tRNA gene expression is coupled to the expression of other chloroplast transcription units.